Ca2+ -independent transmission at the central synapse formed between dorsal root ganglion and dorsal horn neurons

Abstract

A central principle of synaptic transmission is that action potential-induced presynaptic neurotransmitter release occurs exclusively via Ca²⁺ -dependent secretion (CDS). The discovery and mechanistic investigations of Ca²⁺ -independent but voltage-dependent secretion (CiVDS) have demonstrated that the action potential per se is sufficient to trigger neurotransmission in the somata of primary sensory and sympathetic neurons in mammals. One key question remains, however, whether CiVDS contributes to central synaptic transmission. Here, we report, in the central transmission from presynaptic (dorsal root ganglion) to postsynaptic (spinal dorsal horn) neurons in vitro, (i) excitatory postsynaptic currents (EPSCs) are mediated by glutamate transmission through both CiVDS (up to 87%) and CDS; (ii) CiVDS-mediated EPSCs are independent of extracellular and intracellular Ca²⁺ ; (iii) CiVDS is faster than CDS in vesicle recycling with much less short-term depression; (iv) the fusion machinery of CiVDS includes Cav2.2 (voltage sensor) and SNARE (fusion pore). Together, an essential component of activity-induced EPSCs is mediated by CiVDS in a central synapse.

Keywords: Ca2+-dependent secretion; Ca2+-independent but voltage-dependent secretion; dorsal horn; dorsal root ganglion; synaptic transmission.

Figure 1. Ca²⁺‐independent but voltage‐dependent synaptic neurotransmission in co‐cultured DRG–DH neurons

1. A

   (a1) Whole‐cell current recording of evoked EPSC signals in response to local electrical field stimulation (Estim, arrows) from a postsynaptic dorsal horn (DH) neuron co‐cultured with presynaptic dorsal root ganglion (DRG) neurons in Ca²⁺‐free (0Ca²⁺, black) and 2.5 mM Ca²⁺ bath (2Ca²⁺, green); (a2), evoked intracellular Ca²⁺ signals (dF/F₀) in a DRG neuron; inset, micrograph showing the setup for EPSC recording from DH neurons co‐cultured with EGFP‐expressing DRG neurons (scale bar, 20 μm).

2. B

   (b1) Evoked EPSCs from cultured hippocampal neurons in Ca²⁺‐free (black) and 2.5 mM Ca²⁺‐containing solution (green); (b2) evoked intracellular Ca²⁺ signals (dF/F₀) in hippocampal neurons; inset, micrograph showing the setup for EPSC recordings from a hippocampal neuron (scale bar, 20 μm).

3. C

   As in (A), except that 50 μM BAPTA‐AM was pre‐loaded into the DRG and DH neurons before recording in Ca²⁺‐free (black) or 2.5 mM Ca²⁺‐containing solution (green). BAPTA‐AM was loaded for 30 min at 37°C.

4. D

   Left, quantification of amplitude as in (A) (n = 26 cells for 0Ca²⁺ and 29 cells for 2Ca²⁺). Right, quantification of EPSC amplitudes as in (C) (n = 16 cells for 0Ca²⁺ and 14 cells for 2Ca²⁺).

5. E

   Evoked EPSC_CiVDS (0Ca²⁺, black) and total EPSCs (CDS + CiVDS, 2Ca²⁺, green) induced by paired‐pulse stimulation with a 50‐ms interval from DH neurons co‐cultured with DRG neurons. The 0Ca²⁺ and 2Ca²⁺ EPSCs were recorded in two different DH neurons.

6. F

   Summary plot of paired‐pulse ratios as in (E) with different intervals (in 0Ca²⁺, n = 7 cells for 10 ms, 13 for 50 ms, 14 for 500 ms, 10 for 5 s, and 8 for 15 s; in 2Ca²⁺, n = 14 cells for 10 ms, 17 for 50 ms, 22 for 500 ms, 19 for 5 s, and 14 for 15 s). Inset shows the initial plot at an expanded scale.

7. G

   Representative EPSCs induced by a 10‐Hz stimulus train in DH neurons co‐cultured with DRG neurons in Ca²⁺‐free (black) or 2.5 mM Ca²⁺‐containing solution (green). The 0Ca²⁺ and 2Ca²⁺ EPSCs were recorded in two different DH neurons.

8. H

   Summary plots of the EPSC amplitudes as in (G), including CiVDS (0Ca²⁺, black), CDS + CiVDS (2Ca²⁺, green), and CDS (2Ca²⁺ − 0Ca²⁺, blue) (n = 11 cells).

9. I

   As in (H), statistics for amplitudes of EPSC_CiVDS (0Ca²⁺, black) and EPSC_CDS (“2Ca²⁺” − “0Ca²⁺,” blue) evoked by single pulse (first EPSC, left) or 10 pulses (cumulative 10 EPSCs, Σ_EPSC, right) during 10‐Hz train stimulation (n = 11 cells).

Data information: All but (B) were from DRG and DH co‐cultures. EPSC recording and Ca²⁺ imaging were separate experiments. Data are shown as the mean ± s.e.m.; Mann–Whitney test for (D) and (F); paired Student's t‐test for (I); *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant.

Figure 2. Imaging of CiVDS‐mediated synaptic transmission in co‐cultured DRG and DH neurons

1. A

   A representative photograph showing the co‐cultured DRG and DH neurons. The DRG neurons were expressed with Spy‐pHluorin for imaging the synaptic transmission. Scale bar, 10 μm.

2. B

   Images of a presynaptic bouton marked in (A) showing the Spy‐pHluorin fluorescence at 20 s before (−20 s, pre‐stimulus), 20, 40, and 120 s after electrical stimulation (post‐stimulus) in 0Ca²⁺ (upper panel) or 2Ca²⁺ (lower panel) solution.

3. C

   Averaged fluorescence changes (ΔF/F₀) of Spy‐pHluorin in 0Ca²⁺ (left) or 2Ca²⁺ solution (right) in response to the same electrical stimulation (20 Hz, 20 s) (n = 45 puncta from six cells for 0Ca²⁺ and 57 puncta from six cells for 2Ca²⁺). The shadow in the traces represents the error bars (s.e.m) of each point.

4. D–F

   The same as in (A–C), but the experiments were performed in cultured hippocampal neurons (n = 70 puncta from three cells for 0Ca²⁺ and 72 puncta from three cells for 2Ca²⁺).

Figure 3. EPSC_CiVDS is mediated by glutamate transmission

1. A

   Representative evoked EPSCs in DH neurons co‐cultured with DRG neurons before (black) and after (red) applying 50 μM AP5 and 10 μM CNQX in Ca²⁺‐free (left) or 2.5 mM Ca²⁺‐containing solution (right). The EPSCs of 0Ca²⁺ and 2Ca²⁺ were recorded in two different DH neurons.

2. B

   Quantification of EPSC amplitudes as in (A) (n = 15 cells for 0Ca²⁺ and 10 cells for 2Ca²⁺).

3. C

   Evoked EPSCs recorded in DH neurons co‐cultured with DRG neurons before (black) and after (red) applying 100 μM cyclothiazide (CTZ) in 0Ca²⁺ (left) or 2Ca²⁺ solution (right). The traces are fitted with a single exponential curve (blue). The EPSCs of 0Ca²⁺ and 2Ca²⁺ were recorded in two different DH neurons.

4. D

   Quantification of the decay time (τ) and charge as in (C) (n = 10 cells for 0Ca²⁺ and 12 cells for 2Ca²⁺). EPSCs were evoked by local electrical stimulation (Estim) at arrows.

Data information: Data are shown as the mean ± s.e.m.; paired Student's t‐test; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Presynaptic DRG neurons elicit EPSC_CiVDS in postsynaptic DH neurons

1. A

   Setup for paired patch‐clamp recording of dorsal root ganglion (DRG) and dorsal horn (DH) neurons in co‐culture. The presynaptic DRG neuron was whole‐cell dialyzed with 10 mM BAPTA under current‐clamp mode, while the postsynaptic DH neuron was in voltage‐clamp mode.

2. B

   Left, representative dual recordings of presynaptic action potentials (upper) and postsynaptic EPSCs (lower) following current‐step injection (1,000 pA, 5 ms) in a DRG neuron dialyzed with 10 mM BAPTA. Following whole‐cell break‐in and intracellular BAPTA dialysis, double recordings were performed at 0 min/2.5 mM Ca²⁺ bath (green), and 5 min/0Ca²⁺ bath (black), Right, statistics of EPSC amplitudes (n = 9 cells).

3. C, D

   As in (A) and (B), except that EPSCs were recorded from cultured hippocampal neurons (n = 11 cells).

4. E

   Image showing a DRG neuron infected by ChR2‐mCherry AAV2/9 virus and a co‐cultured DH neuron.

5. F

   Upper, diagram showing the protocol for infection of DRG neurons by ChR2‐mCherry virus and co‐culture with DH neurons; lower, cartoon of EPSC recording from DH neurons co‐cultured with ChR2‐expressing DRG neurons.

6. G

   Left, typical EPSCs from DH neurons induced by light stimulation (475 nm, 5 ms, at arrows) of co‐cultured ChR2‐expressing DRG neurons in Ca²⁺‐free or 2.5 mM Ca²⁺‐containing solution before (black) and after (red) exposure to 50 μM AP5 and 10 μM CNQX. The EPSCs of 0Ca²⁺ and 2Ca²⁺ were recorded in two different DH neurons; right, statistics of EPSC amplitude (n = 5 cells for 0Ca²⁺ and 14 for 2Ca²⁺).

Data information: Data are shown as the mean ± s.e.m.; Wilcoxon test for (B); paired Student's t‐test for (D) and (G); **P < 0.01, ***P < 0.001. Scale bars, 20 μm.

Figure 5. EPSC_CiVDS are mediated by the SNARE complex and N‐type Ca²⁺‐channels

1. A

   Left, diagram showing the protocol for EPSC recording from DH neurons co‐cultured with Syb2‐knockout (cKO) DRG neurons; right, cartoon of EPSC recording from DH neurons co‐cultured with Syb2‐cKO DRG neurons.

2. B

   Representative western blots (upper) and analysis (lower) of the expression levels of Syb2 in control (Ctrl) and Syb2‐cKO DRG neurons (n = 3 per group). Control DRG neurons are from homozygous floxed Syb2‐null mice infected with AAV5‐CAG‐EGFP virus.

3. C

   Evoked EPSCs and statistics from DH neurons co‐cultured with control (Ctrl) or Syb2‐cKO DRG cells (cKO) in 0Ca²⁺ solution (n = 13 cells for Ctrl and 11 for cKO).

4. D

   Evoked EPSCs and statistics from DH neurons co‐cultured with DRG neurons before (black) and after (red) applying 1 μM ω‐conotoxin‐GVIA (GVIA) in 0Ca²⁺ bath (n = 9 cells).

5. E

   Upper, diagram showing the protocol for EPSC recordings from DH neurons co‐cultured with Cav2.2 knockdown (KD) DRG neurons; lower, cartoon of EPSC recording from DH neurons co‐cultured with Cav2.2‐KD DRG neurons.

6. F

   Left panels, evoked EPSCs from DH neurons co‐cultured with scrambled shRNA control (Ctrl) or Cav2.2‐KD (sh‐1/sh‐2) DRG neurons in 0Ca²⁺ solution. Right panel, quantification of evoked EPSCs (n = 17 cells for Ctrl, 12 for sh‐1, and 19 for sh‐2). EPSCs were evoked by local electrical stimulation (Estim, at arrows).

Data information: Data are shown as the mean ± s.e.m.; paired Student's t‐test for (B) and (D), unpaired Student's t‐test for (C), and Kruskal–Wallis test followed by Dunn's multiple comparisons test for (F); *P < 0.05, **P < 0.01, ***P < 0.001. Source data are available online for this figure.

Figure 6. Model of the CiVDS‐mediated EPSC in synaptic transmission

In Ca²⁺‐free bath, an action potential activates the voltage‐gated Ca²⁺ channel (VGCC/Cav2.2) and triggers presynaptic glutamate release through Ca²⁺‐independent but voltage‐dependent secretion (CiVDS) and activates an excitatory postsynaptic current (EPSC_CiVDS). In physiological solution containing Ca²⁺, however, both CiVDS‐ and Ca²⁺‐dependent secretion (CDS)‐mediated glutamate release contribute to a larger evoked postsynaptic EPSC (not shown).